A scientific search for therapeutically useful compounds in combating psychostimulant abuse and addiction has been in progress for decades, with no clinically available agents to date. A major hindrance toward this goal has been the limited information on the structure and function of the plasma membrane monoamine transporter proteins. Pharmacological and behavioral studies indicate that the dopamine transporter protein (DAT) is the brain receptor chiefly responsible for the reward/reinforcing properties of cocaine and the amphetamines. The serotonin transporter protein (SERT) also plays a key role in the mood-altering effects of these drugs, especially in the case of amphetamines such as MDMA ("ecstasy"). The DAT and SERT are responsible for termination of neurotransmission by uptake of the monoamine neurotransmitter from the neuronal synapse. The long-term objective of this application is to understand how cocaine and the amphetamines interact with the DAT and SERT, which entails mapping the DAT and SERT binding pockets of these drugs at the level of the amino acid residue. This level of resolution may now be a possibility, as the recently published X-ray crystal structure of the LeuTAa transporter protein, homologous in sequence to the DAT and SERT, can serve as a template for creation of 3-dimensional DAT and SERT computer models. The two specific aims of this proposal are to create LeuTAa-based 3-D DAT and SERT models, and use the models to identify and characterize via site-directed mutagenesis and pharmacology the DAT and SERT residues key in substrate (e.g., dopamine, serotonin, various amphetamines) and inhibitor (e.g., cocaine, methylphenidate) recognition. The resultant pharmacologic data will be used to refine the molecular models to the point that they can be employed in in silico screening of structural libraries containing hundreds of thousands of "small molecule" compounds in the search for novel DAT and SERT ligands. These specific aims will be addressed by DAT and SERT computer-aided molecular modeling to identify logical mutagenesis targets, site-directed mutagenesis of the cDNAs encoding the DAT and SERT proteins, transfecting mammalian cell lines with wildtype or mutant DAT or SERT cDNAs, assaying transfected cells for binding and uptake of radiolabeled DAT or SERT ligands, inhibition of radioligand binding or uptake with non-radioactive competing DAT or SERT ligands, and assessment of cell surface expression of the transporter proteins via immunoblotting techniques. Results from the proposed experiments should reveal specific insights as to how cocaine and the amphetamines exert their effects via these monoamine transporters, as well as information on DAT recognition of drugs with less abuse potential such as benztropine and methylphenidate and SERT recognition of antidepressant drugs. Elucidating at the amino acid residue level the mechanisms of discriminating abused and non-abused substrates and inhibitors should provide a blueprint for rational design of medications that block cocaine and amphetamine actions without in turn carrying the potential for abuse and addiction. Additionally, these studies may lead to therapeutics for other DAT- or SERT-related conditions including depression, anxiety disorders, attention deficit hyperactivity disorder, migraine, narcolepsy and Parkinson's disease. The proposed dopamine transporter (DAT) and serotonin transporter (SERT) studies tease apart binding requirements for psychostimulants of low abuse potential (benztropine and methylphenidate) and high abuse potential (cocaine and methamphetamine), and should even allow discrimination of monoamine transporter recognition mechanisms for cocaine, methamphetamine and MDMA ("ecstasy"). Exploring exactly how specific DAT and SERT protein components contribute to recognition of abused and non-abused drugs should provide a blueprint for designing medications that block actions of cocaine and the amphetamines without in turn carrying the potential for abuse and addiction. Additionally, these studies may lead to therapeutics for other DAT- or SERT-related conditions including depression, anxiety disorders, attention deficit hyperactivity disorder, migraine, narcolepsy and Parkinson's disease. [unreadable] [unreadable]